Antibodies are important tools for medical applications. Most antibodies are composed of two heavy and two light chains, both chains form the antigen binding site. Non-conventional antibody structures have been found in llamas, camels, and cartilaginous fishes. These antibodies consist of a single heavy chain with four constant domains and an antigen binding site or variable domain denominated VHH, hcAbs in camels and vNAR or VHNAR in elasmobranches.1 
Antibody technology has been developed to provide new therapies and diagnostic systems. It includes, for example, the use of monoclonal antibodies; humanized antibodies, designed to decrease the non-human antigen response; and conjugated antibodies, to improve their properties. The number of antibodies approved by FDA for the treatment of several human diseases has been increasing, approximately 352 of them are on clinical trials (phase I and phase II), accounting for around 25% of all the proteins on clinical trials. A lot of effort has been done in order to reduce the conventional size of antibodies and preserving their antigen binding properties like affinity, avidity and specificity.2 
Small fragments of antibodies with antigen binding ability are among the technologic alternatives for medical use. Such alternatives have progressed from recombinant molecules, like the fragment of antigen binding (Fab) and/or the single chain variable fragment (scFv), to single binding domains for proteins based on immunoglobulins with VH domains, which in turn have been used to develop new immunotherapeutic and immunodiagnostic strategies. Mimetics of the Fab's to smaller domains is advantageous since that increases the stability and the possibility for accessing antigenic epitopes that are not recognized by conventional antibodies.2 
There are three isotypes of immunoglobulins or antibodies from cartilaginous fishes, two of them with two standard heavy and light chains, designated as IgM and IgW (also called IgX or IgNARC) and one atypical isotope called IgNAR which is a homodimer of heavy chains not associated with light chains. The shark antigen receptor immunoglobulins (referred as IgNAR or NAR) have a single variable domain (sdAb fragments) and two fork hypervariable structures to include the entire repertoire with union specificity to recognize the antigens. IgNARs are high soluble and high thermostable small molecules (12 kDa) and with good tissue penetration in vivo, which makes IgNARs a good resource for antibody engineering and therapeutic antibodies.3,4 
The present invention concerns to selection and isolation of IgNAR antibodies, in particular of its variable region VHNAR, originated in the immunized shark Heterodontus francisci or Orectolobus maculatus with affinity for cytokines and ability to neutralize their activity. These antibodies are originated generally by the immunological system of cartilaginous fish (sharks, skates, rays, and chimeras). The molecular arrangement of the IgNAR antibodies consists of five constant regions and one variable region which in addition is very similar to the VH found in camelids, which possibly represents an evolutionary convergence at molecular level.1,5 
Nuttall and collaborators obtained a non-immune shark antibody library, through phage display technology based on variable regions of IgNAR of the shark Orectolobus maculatus. These regions have the ability to recognize proteins like gingipain K protease from Porphyromonas gingivalis, the mitochondrial import receptor Tom70, the lysozyme and the Apical Membrane Antigen 1 (AMA1) of Plasmodium falciparum, among others. These regions have been cloned in Escherichia coli expression systems, being the first description of antigenic specificity of NARs obtained from the natural repertoire of the shark as a probable source of high affinity single domain antibodies.6,7,8 
Dooley and collaborators in 2003 selected a targeted library generated in Ginglymostoma cirratum. These sharks were immunized with hen egg lysozyme (HEL), resulting in highly specific clones to HEL antigen, with a nanomolar affinity (ranging from 10−7 to 10−10 M) and with a great resistance to heat denaturalization, since they maintained more than 20% of its activity after 3 hrs of incubation at 100° C.9 
The genes of IgNAR are grouped; each group consists of a single variable simple region (VH), three elements of diversity (D) and a single joining gene (J). The primary repertoire of IgNAR VH is generated by four recombination events, resulting in a diverse repertoire of CDR3 both in terms of sequence and length.6 
Different technologies with shark proteins were developed immediately after the discovery of these single chain antigen receptors due to their high functionality. The isolated and cloned variable domain is very stable; it is 20% smaller than the domain of camelid antibodies and it possesses the same antigen binding ability than the original receptor.
The advantage of this technology is that it combines the properties of the conventional antibodies with the advantages of the small molecules; they have high specificity and low inherent toxicity; due to their low molecular weight they have more possibilities to reach their target site; they are capable of inhibiting enzymes and they can also reach the binding site of cell receptors. All these properties can be exploited for therapeutic uses. Additionally, they have a great potential for being administered by diverse routes, including the topical route. Finally, their production is easy and at low cost.5 
From the literature it is clear that overexpression of VEGF and their receptors (VEGFR-1, VEGFR-2 and VEGFR-3) is causing increased microvascular permeability and angiogenesis, producing eye pathologies such as diabetic retinopathy, age-related macular degeneration (ARMD), and neovascular glaucoma. The cellular distribution of VEGFR-1, VEGFR-2 and VEGFR-3 receptors suggests various specific functions of the VEGF family in normal retina, both in the retinal vasculature and in neuronal elements.10 
The vascular endothelial growth factor (VEGF) has been described as a tumor-derived factor with the ability to induce endothelial cell permeability, cell proliferation and angiogenesis, which defines formation of new blood vessels, especially those providing oxygen and nutrients to cancerous tissues. Although many other factors are involved in angiogenesis, VEGF is the key mediator.
The VEGF (or VEGF-A) is a heparin-binding glycoprotein that belongs to a subfamily of growth factors that includes VEGF-B, VEGF-C, VEGF-D and platelet growth factor. As a result of alternative splicing patterns of VEGF mRNA, VEGF exists in at least seven isoforms. The four major isoforms are VEGF121, VEGF165, VEGF189 and VEGF206 (the subscripts refer to the number of amino acids of the protein). The predominant species is VEGF165 with an affinity for heparin; therefore, part of this isoform is bound and is released by proteolytic cleavage. The rest is free and available for binding to receptors on endothelial cells and it is the result of two distinct processes: the secretion of soluble isoforms and the proteolytic cleavage of bound isoforms. The physiological importance of the different isoforms of VEGF is not clear; however the VEGF165 is the major regulator of angiogenesis.
The VEGF binds mainly to two receptors: VEGF receptor-1 (also known as Flt-1) and VEGF receptor-2 (also known as Flk-1 or KDR). Each of these receptors has an extracellular domain (which binds VEGF) with seven immunoglobulin-type areas, a single transmembrane region and an intracellular domain with tyrosine kinase activity. These receptors are mainly found in vascular endothelial cells of developing tissues.
Binding to VEGF receptor-2, directly stimulates angiogenesis and activates a series of signal transduction pathways resulting in the proliferation of vascular endothelial cells, migration of vascular endothelial cells, survival of immature endothelial cells and increased vascular permeability.
Although VEGF receptor-1 was initially thought to act as a “decoy receptor” by reducing the number of molecules of VEGF capable of binding to VEGF receptor-2, recent studies show that VEGF receptor-1 is also capable of inducing a mitogenic signal.
Angiogenesis is the formation of new vascular structures and plays a key role in pathological processes such as the establishment of tumors and eye diseases. Diabetic retinopathy is known as the abnormal growth of new blood vessels and the appearance of fibrous tissue in the retina; when originating beneath the macula it is called Macular Degeneration; and when it is occurs in the iris it is called Neovascular Glaucoma.
Diabetic retinopathy is a condition of the retina that occurs in patients with diabetes mellitus; both type 1 and type 2 after several years of having the disease, especially when the disease is not well controlled. There are two types of diabetic retinopathy: early or non-proliferative diabetic retinopathy and proliferative or advanced diabetic retinopathy. The proliferative diabetic retinopathy is characterized by the abnormal growth of new vessels and subsequent fibrous proliferation in response to retinal ischemia as well as the development of pre-retinal or vitreous hemorrhage.11 Its importance lies in the fact that it is one of the leading causes of irreversible blindness worldwide and that it can be prevented by taking the proper precautions and applying timely treatment.11 Diabetic retinopathy is defined as the presence and evolution of typical ocular microvascular injuries in diabetic patients.
Age-related macular degeneration is the leading cause of visual loss in patients over 60 years. The macula is the central area of the retina, and it is responsible of the fine vision used for reading, watching television, see the factions of people and in general the vision of any fine details.12 The ARMD is a degenerative condition of the macula, which is a common cause of vision loss. It can be classified as wet (neovascular) or dry (non-neovascular). About 10% of the patients suffer from wet macular degeneration. Usual treatment of wet macular degeneration involves the application of one or several injections of medicines within the eye called “antiangiogenics”, whose intention is to remove the neovascular membrane. With this treatment, over 90% of patients achieved to maintain vision, and approximately two thirds of patients improved vision, as long as the treatment is applied in a timely manner and not much scarring occurs.
There is the development of new blood vessels in those tissues where the circulation is either damaged through trauma or disease such as those mentioned above. Corneal neovascularization is the abnormal growth of blood vessels causing choriocapillaries passing through Bruch's membrane and then proliferate under the retinal pigment epithelium (type 1) and/or under the retina (type 2). This can occur by rupture of Bruch's membrane, the release of cytokines such as VEGF, inflammation, oxidative stress in the retinal pigment epithelium or vascular insufficiency. This condition is the leading cause of wet macular degeneration and may be associated with various disorders including angioid streaks, choroidal rupture, pathological myopia, chorioretinal lesion and birdshot chorioretinopathy.
There is also the phenomenon of iris neovascularization. The abnormal formation of new blood vessels on the anterior surface of the iris is commonly associated with different conditions which have led to retinal ischemia, such as diabetic retinopathy, central retinal vein occlusion, carotid artery disease, melanoma uveal, prolonged retinal detachment, etc. Neovascularization begins in the pupil margins and often at the same time at the angle of the anterior chamber and spreading over the entire surface. The new vessels are associated with fibrous tissue membranes, which can block the pass of aqueous humor through the trabecular meshwork (neovascular glaucoma) and cause ectropion uveae in the pupillary border. Its usual treatment consists of applying laser photocoagulation to prevent the formation of new blood vessels.
Neovascular glaucoma is a special type of secondary glaucoma occurring as a consequence of the formation of new blood vessels in the iris. These new vessels eventually cause a blockage in the circulation of aqueous humor from the anterior chamber of the eye, which triggers an ocular hypertension. It results from a lack of chronic and maintained retinal oxygen. In response thereto the system produces a number of substances that stimulate neovascularization.
Other pathological processes where the phenomenon of neovascularization is involved are: Retinal Neovascularization, Choroidal Neovascularization, Corneal Neovascularization, Macular Degeneration, Age-Related Macular Degeneration, Retinal Diseases, Diabetic Retinopathy, Vitreous Hemorrhage, Retinal Hemorrhage, Choroiditis, Retinal Detachment, Retinal Drusen, Neovascular Glaucoma, Choroid Diseases, Uveitis, Myopia, Eye Diseases, Fungal Eye Infections, Telangiectasia, Retinal Artery Occlusion, Degenerative Myopia, Retinal Vein Occlusion, Chorioretinitis, Histoplasmosis, Uveal Diseases, Rubella (German Measles), Ocular Toxoplasmosis, Epiretinal Membrane, Coloboma, Choroid Neoplasms, Retinal Degeneration, Retinitis, Retinal Perforations, Herpetic Keratitis, Retinopathy of Prematurity, Cystoid Macular Edema, Papilledema, Uveomeningoencephalitic Syndrome, Optic Disk Drusen, Angioid Streaks, Retinitis Pigmentosa, Vision Disorders, Sympathetic Ophthalmia, Scar, Ocular Burns, Recurrent Ischemia, Eye Injuries, Glaucoma, Eye Hemorrhage, Scotoma, Posterior Uveitis, Fungemia, Retinal Neoplasms, Corneal Diseases, Pigmentary Incontinence, Hemoglobin C Disease, Fibrosis, Opacity of the Cornea, Anterior Uveitis, Hyphema, Sarcoidosis, Aphakia, latrogenic Disease, Panuveitis, Eye Cataract, Postoperative Complications, Sickle Cell Anemia, Retinal Vasculitis, Osteoma, Cytomegalovirus Retinitis, Atrophy, Phlebitis, Keratoconus, Sturge-Weber Syndrome, Viral Eye Infections, Eye Abnormalities, Substance-Related Disorders, Penetrating Eye Injuries, Diabetes Mellitus Type 2, Radiation Injuries, Sickle Cell Trait, Pseudophakia, Pigmented Nevus, Proliferative Vitreoretinopathy, Bleeding, Diabetes Mellitus Type 1, Nevus, Optic Nerve Diseases, Vascular Diseases, Candidiasis, Chemical Burns, Microphthalmia.
Worldwide, 285 million people have visual impairment from various causes, and 39 million of them are blind.14 “The main causes of chronic blindness include cataract, glaucoma, age-related macular degeneration, corneal opacities, diabetic retinopathy, trachoma and eye conditions in children as well as those caused by lack of vitamin A. The age-related blindness as well as due to uncontrolled diabetes is increasing worldwide. Three quarters of all blindness cases are preventable or treatable”.15 
The inhibitory molecules of VEGF activity may be used to limit neovascularization processes which depend upon VEGF action.
The anti-VEGF antibodies bind to the ligand, thus eliminating free-circulating VEGF and preventing its binding to its receptors. Antibodies have been used for this purpose since they are highly specific and only bind to VEGF; the pro-angiogenic effects mediated by all receptors binding to VEGF can be inhibited. Different strategies have been developed to inhibit VEGF-mediated signaling, however, since it showed that a specific anti-VEGF antibody could inhibit tumor growth in animal models described by Ferrara and Davis-Smith, in 1997 began the development of a human version of anti-VEGF antibody.
Bevacizumab is an anti-VEGF monoclonal antibody. This has been the first anti-angiogenic agent approved for cancer treatment; it has been approved for use as a first-line treatment of metastatic colorectal cancer in combination with a chemotherapy regimen. It has been tested in cancers of many organs with positive clinical outcomes including tumor regression and increased medium to long-term survival rate.16 
In 2004, the FDA accepted the Pegaptanib, the first antiangiogenic drug for the eye administered by intravitreal injection. This anti-VEGF was analyzed in studies of patients with age-related macular degeneration. The results showed stabilization of vision in 70% of treated patients, versus 50% in patients not treated with this antibody.
In 2006, the FDA approved the use of ophthalmic Ranibizumab, which is a recombinant Fab fragment of anti-VEGF humanized murine monoclonal antibody; it has also been used successfully in the treatment of eye diseases for the inhibition of neovascularization that leads to blindness, especially for treating macular degeneration in all its forms, particularly wet ARMD.17 
The application route of Ranibizumab is intravitreal injection. However, retinal detachment and serious infections are among the side effects caused by Ranibizumab. It has been reported that in mouse it causes the death of photoreceptors and Müller cells of the retina, which are essential for visual function.
Other ophthalmic drugs that act by inhibiting the activity of the VEGF, and intraocularly administered, are the following: the Verteporfin, used as a selective treatment of choroidal neovascularization associated with macular degeneration; Aflibercept which is used to treat wet age-related macular degeneration, and dexamethasone, corticosteroid which has shown to reduce the inflammatory process causing the macular edema when applied as an intravitreal implant.
U.S. Pat. No. 8,496,933, Paniagua-Solis et al., refers to the selection, isolation and production of a protein belonging to variable regions named VHNAR or vNAR, originated from IgNAR-type immunoglobulins of elasmobranches with antigen receptor abilities. This vNAR was named V13 and it was selected by its capacity to bind specifically to the vascular endothelial growth factor (VEGF). It works by neutralizing the activity of VEGF, and it has been characterized by its sequence, selected and optimized, and which is the closest state of the art to the invention, incorporated herein by reference in its entirety.
The trials on anti-VEGF therapies have tried a variety of dosing strategies such as: when to start treatment, dosing frequency, and how these strategies can be followed in medical treatment, since secondary or side effects as hypertension, proteinuria, bleeding, damage to the healing of surgical wounds, even fatal complications such as arterial thrombosis, gastrointestinal perforation and reversible posterior focal leukoencephalopathy, route of administration, the invasiveness of the methods, the high dose, bioavailability, instability as well as high costs, long treatments, among others, lead to the need of research for new molecules that have better performance. Even with such alternatives, it is required to develop better drugs that inhibit the activity of VEGF for eye treatments in order to remove or reduce side effects.
The present invention describes novel clones and molecules named V19, V32R and the aforementioned V13, characterized by their three-dimensional structure, their sequences and affinities to VEGF and useful in treating eye conditions, particularly for the treatment of diabetic retinopathy, macular degeneration, neovascular glaucoma or ocular conditions related to angiogenesis.